1. Field of the Invention
Osteoporosis is one of the most widespread diseases. This disorder is characterized by a decrease in bone mass leading to a weakening of the bone. The degree of weakening can be severe enough to result in fractures occurring spontaneously or a result of minor trauma.
There is a continuing need to develop therapeutics for the management of osteoporosis and other metabolic bone diseases. In order to develop and evaluate such drugs, a convenient and accurate means by which bone turnover can be monitored must be available. Currently, the diagnosis of osteoporosis depends on radiological and densitometric monitoring, bone biopsy examination and the assaying of bone specific biochemical markers.
Bone formation is monitored in serum through the use of biochemical markers by various assays including those for alkaline phosphatase, osteocalcin (bone Gla protein) or levels of the terminal extensions of procollagen.
Although the function of bone alkaline phosphatase ("B-ALP") in vivo is unknown, this isoenzyme is thought to be involved in bone formation. Several lines of evidence suggest that measurement of B-ALP activity in serum might prove a useful index of the bone formation rate:
(1) B-ALP is localized in the plasma membranes of osteoblasts, which are the bone-forming cells. PA1 (2) In vitro studies have shown that the amount of alkaline phosphatase activity in fetal rat calvaria is proportional to the rate of collagen production. Canalis, Metabolism 32:14-20 (1983) PA1 (3) In vivo studies with normal young mice have shown a correlation between serum alkaline phosphatase activity and osteoblast number. Marie, et al., Calcif Tissue Int 35:418-425 (1983) PA1 (4) The amount of B-ALP activity in human serum is proportional to the rate of bone formation, at least when both are elevated as in young children and patients with Paget's disease. Farley, et al., Metabolism 35:563-571 (1986).
Since there is a critical demand for non-invasive measurements of bone formation and resorption rates, there is a need for a method of measuring B-ALP in serum as an index of the bone formation rate.
Ordinarily, human serum contains a variable mixture of related forms of alkaline phosphatase isoenzymes that can interfere with accurate assay results. These isoenzymes are derived from bone, kidney, intestinal, liver and placental tissues. Four different genes code for the main groups of alkaline phosphatase isoenzymes. Although the isoenzymes derived from the liver, kidney, and bone are the same gene product, they differ from each other on the basis of electrophoretic mobility and heat and urea stability. These differences are thought to be due to a post translational modification such as glycosylation.
One method of attempting to distinguish between the isoenzymes has been the use of monoclonal antibodies. However, most attempts to produce antibodies specific for B-ALP over liver alkaline phosphatase have resulted in cross reactive antibodies.
The present invention is directed to the monitoring of bone formation through the use of bone alkaline phosphatase.
More particularly, this invention concerns a method for determining the presence or amount of B-ALP in a sample using monoclonal antibodies that preferentially recognize B-ALP over the liver isoenzyme.
2. Description of the Related Art
Singh, et al., Experimental Cell Research 95:347-358 (1975) pertains to the use of antibodies to show that alkaline phosphatase from bone ("B-ALP") is a specific enzyme distinct from alkaline phosphatases from other sources.
Bailyes, et al, Biochem. J. 244:725-733 (1987) describes the preparation of B-ALP and liver alkaline phosphatase ("L-ALP") antibodies and their use in immunoaffinity purification of the enzymes.
Mulivor, et al., J. Lab. Clin. Med. 105(3):342-347 (1985) pertains to the use of monoclonal antibodies in immunoprecipitation of liver/bone/kidney, placental and intestinal alkaline phosphatase.
Hill, et al., European Patent Application No. 0381450 and Clinica Chimica Acta 186:315-320 (1989) pertain to monoclonal antibodies specific for B-ALP and not L-ALP, where one of the antibodies (BA1G 121) showed &lt;3% cross-reactivity with L-ALP.
Hill, et al., Journal of Bone and Mineral Research 6:S244 (1991) describes an immunoradiometric assay for monitoring bone metastasis using monoclonal antibodies that are capable of distinguishing B-ALP from L-ALP.
Anderson, et al., U.S. Pat. No. 5,087,573 pertains to monoclonal antibodies that distinguish B-ALP from liver or kidney alkaline phosphatase.
Masuhara, et al., International Orthopaedics 15:61-64 (1991) describes an antibody (1B3.7) having about 3.5 fold greater binding to B-ALP than to L-ALP.
Seabrook, et al. Clinica Chimica Acta 172:261-266 (1988) pertains to a monoclonal antibody that exhibits 1.8 fold preference for B-ALP versus L-ALP.
Nagoya, et al. Jpn. J. Cancer Research 82:862-870 (1991) describes the detection of B-ALP by monoclonal antibodies that react with human osteosarcoma-associated antigen.
The art also describes antibodies that are specific to L-ALP over B-ALP. For example, Lawson, et al., Clinical Chemistry 31(3):381-385 (1985) pertains to a monoclonal antibody that exhibits a five-fold greater response with L-ALP than with B-ALP.
There are numerous references that pertain to assays for analytes with more than one epitope, in particular, assays that use two antibodies in a sandwich format. The following are illustrative of the state of the art.
Jeong, et al., U.S. Pat. No. 4,244,940 describes a sandwich assay useful for any ligand that can simultaneously become bound by two receptors.
Bunting, U.S. Pat. No. 4,271,140 pertains to a double receptor assay using a receptor complex comprised of a binding ligand, a receptor for the binding ligand and a receptor for the analyte.
Murad, et al., U.S. Pat. No. 4,474,892 describes a sandwich assay using two antibodies that are of a different class or subclass.
Forrest, et al., U.S. Pat. No. 4,659,678 pertains to an assay for an antigen with multiple epitopes using a reagent consisting of an antibody that is conjugated to a hapten or antigenic substance, where an antibody that binds the reagent, is bound to a support.
Schurrs, et al., U.S. Pat. Reissue No. 32,696 describes a sandwich assay where one reactant is bound to a support and one reactant is bound to an enzyme;
Zahradnik, et al., U.S. Pat. No. 4,935,339 pertains to an assay using a first monoclonal antibody, and a second different monoclonal antibody or a polyclonal antibody of restricted specificity, and a high affinity ligand for separation.
Gallati, et al., U.K. Patent Application No. 2,074,727 describes an assay for an antigen using two antibodies from different clones, which are directed towards different epitopes of the antigen.
Tanaka, et al., U.K. Patent Application No. 2,190,490 pertains to an assay using two antibodies that are from different animal species.